A practical non-enzymatic urea sensor based on NiCo2O4 nanoneedles

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A practical non-enzymatic urea sensor based on

NiCo

2 O

4 nanoneedles†

Sidra Amin,abcAneela Tahira, aAmber Solangi,bValerio Beni, eJ. R. Morante,d

Xianjie Liu,fMats Falhman,fRaﬀaello Mazzaro,aZafar Hussain Ibupoto *ag

and Alberto Vomiero *a

We propose a new facile electrochemical sensing platform for determination of urea, based on a glassy carbon electrode (GCE) modiﬁed with nickel cobalt oxide (NiCo2O4) nanoneedles. These nanoneedles

are used for theﬁrst time for highly sensitive determination of urea with the lowest detection limit (1 mM) ever reported for the non-enzymatic approach. The nanoneedles were grown through a simple and low-temperature aqueous chemical method. We characterized the structural and morphological properties of the NiCo2O4 nanoneedles by TEM, SEM, XPS and XRD. The bimetallic

nickel cobalt oxide exhibits nanoneedle morphology, which results from the self-assembly of nanoparticles. The NiCo2O4 nanoneedles are exclusively composed of Ni, Co, and O and exhibit

a cubic crystalline phase. Cyclic voltammetry was used to study the enhanced electrochemical properties of a NiCo2O4nanoneedle-modiﬁed GCE by overcoming the typical poor conductivity of

bare NiO and Co3O4. The GCE-modiﬁed electrode is highly sensitive towards urea, with a linear

response (R2¼ 0.99) over the concentration range 0.01–5 mM and with a detection limit of 1.0 mM. The proposed non-enzymatic urea sensor is highly selective even in the presence of common interferents such as glucose, uric acid, and ascorbic acid. This new urea sensor has good viability for urea analysis in urine samples and can represent a signi_{ficant advancement in the field, owing to the} simple and cost-effective fabrication of electrodes, which can be used as a promising analytical tool for urea estimation.

Introduction

Urea (also known as carbamide, carbonyl diamide) is a biomolecule and the majornal metabolite of nitrogenous compounds in living organisms, accounting for 70–80% of nitrogen excretion in humans. Urea is present in urine or blood and is deeply investigated in clinical studies. It is extensively used as fertilizer and present in a variety of food products. Therefore its selective and sensitive monitoring through cost

eﬀective methodologies is very important.1_{In the human body,}

urea is formed exclusively in the liver, and it is transported by the bloodstream to the kidneys, where it is excreted into the urine,2 _{as an end product of protein metabolism.}3 _Several

methods have been developed for the detection of small biomolecules using nanostructure materials4–6

To date several analytical methods have been proposed for the determination of urea, such as high performance liquid chromatography (HPLC),7,8 _{gas chromatography (GC)}9_,14

N-NMR,10 _{infrared (IR) spectrometry,}11,12 _calorimetry,13

uorim-etry,14 _{and chemiluminescence.}15 _{All these methods for the}

determination of urea are either costly or require expertise in handling the instruments and are not suitable for eld measurements.16,17_{Another strategy for urea determination is}

through electrochemical sensing/biosensing, by potentiometric measurements in the conguration of ion selective electrode and ammonium ion-generating enzyme,18 _{where poly(vinyl}

alcohol) containing styrylpyridinium PVA/SbQ membrane19_are

commonly employed. However, potentiometric urea biosensors have intrinsic limitations such as high interference with competing species such as uric acid, Na+_{, K}+ _{ions, etc., high}

detection limit and slow response time (see Table 1). Addi-tionally, urea biosensors based on copolymer indium-tin-oxide

aDivision of Materials Science, Department of Engineering & Mathematics, Lule˚a

University of Technology, 97187 Lule˚a, Sweden. E-mail: Alberto.vomiero@ltu.se; zafar.ibupoto@ltu.se

b_{National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro}

76080, Pakistan

c_{Department of Chemistry, Shaheed Benazir Bhutto University, Shaheed Benazirabad}

67450, Sindh, Pakistan

d

Catalonia Institute for Energy Research (IREC), Jardins de les Dones de Negre 1, Sant Adri`a del Bes`os, Barcelona 08930, Catalonia, Spain

e_{RISE Acreo, Research Institute of Sweden, Norrk¨}_{oping, Sweden}

f_{Department of Physics, Chemistry and Biology, Surface Physics and Chemistry,}

Link¨oping University, Faculty of Science & Engineering, Sweden

g_{Institute of Chemistry, University of Sindh, Jamshoro 76080, Sindh, Pakistan}

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9ra00909d

Cite this: RSC Adv., 2019, 9, 14443

Received 2nd February 2019 Accepted 14th April 2019 DOI: 10.1039/c9ra00909d rsc.li/rsc-advances

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Open Access Article. Published on 08 May 2019. Downloaded on 11/5/2019 10:28:04 AM.

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(ITO),3 CH–Fe

3O4/TiO2,20electrochemical hydrolase-based and

creatinine biosensors21_{involve complicated steps in the}

fabri-cation of electrode, which are not economically feasible and environmental friendly.

Therefore, it is desired to develop a user-friendly method for direct in eld urea measurement. Suitably designed nano-materials can oﬀer specic functionalities for the purpose. The catalytic, electrochemical and electric properties of nano-structured oxide materials strongly depend on their size, shape and surface morphology.22–26 Specically, the electronic and surface properties of 1- and 2-dimensional nanostructures, such as large specic surface area, mechanical stability, low potential either for oxidation or reduction, light weight, fast electron transport are useful for the development of advanced electro-chemical biosensors and sensors.27,28_{Non-enzymatic biosensors}

with metal-based catalysts have been widely used due to their high stability and enhanced sensitivity. Transition metal oxides such as CuO, ZnO, and NiO have been widely used for the determination of urea.29 _{However, these nanostructures used}

urease enzyme and exhibit poor performance, not allowing their practical applications. In particular, the drawback of NiO nanostructures used in the non-enzymatic urea senor is its poor conductivity, limiting the response to a narrow range of urea concentration and high detection limit.30_{The bottleneck in CuO}

1-D sensors is that it is a very challenging task to get abundant CuO nanowires by low temperature aqueous chemical growth methods, thus limiting the possibility of scaling up. ZnO nanostructures exhibit poor catalytic properties that limits its wide spread applications in non-enzymatic technology for the detection of various analytes. For these reasons, the investiga-tion of a new class of sensors is highly welcome. The use of bimetallic oxide nanostructure can help by mitigating the issue of poor electrical conductivity and slow charge transfer processes. They demonstrated of great interest for the devel-opment of future devices, like, for instance, in supercapacitors due to their fast charge transfer kinetics. Among them, nickel cobalt oxide (NiCo2O4) exhibits electron conductivity two orders

of magnitude larger than pure NiO or Co3O4. Therefore, it has

been used in diverse applications.31–33 However, there is no report on non-enzymatic urea sensor based on 1-D metal oxides,

which deserve to be investigated owing to their outstanding physical and electronic properties of these nanostructures.

In the present work, we demonstrated for therst time the electrochemical determination of urea by using NiCo2O4

nanoneedle-modied glassy carbon electrode (GCE). Electro-analytical detection based on the use of nanoneedle-modied GCE for urea sensing can oﬀer a practically viable method, free from sample pretreatment, long analysis time and sophis-ticated experimental setup. The modied electrode was also used for the quantication of urea from real samples.

These results oﬀer a new opportunity towards the develop-ment of non-enzymatic urea sensors for clinical analyses and hospitals, in which highly sensitivities and broad range of linear response for urea detection are required. Further development includes the tuning of the nanostructure morphology or the use of three-dimensional porous supporting material for the growth of 1-D hierarchical nanostructures.

Experimental

The scheme of production of the new electrode and its appli-cation as urea sensor is illustrated in Fig. 1, from the synthesis of the oxide nanomaterials, to the test as non-enzymatic urea sensor.

Reagents and solutions

Cobalt chloride hexahydrate (CoCl2$6H2O), nickel chloride

hexahydrate (NiCl2$6H2O), urea (CH4N2O), were purchased

from Sigma-Aldrich (Stockholm, Sweden) and used without any further purication. All chemicals were of analytical grade reagents and were used as received.

All glassware was washed by soaking in 3 M HNO3overnight

followed by washing with detergent water. It was then thor-oughly washed with tap water andnally rinsed at least 3 times with doubly distilled water. The glassware was then dried in an oven at 110C.

Synthesis of NiCo2O4nanoneedles and fabrication on glassy

carbon electrode

Aqueous chemical growth method at low temperature has been applied for the development of NiCo2O4 nanostructures. For

this purpose, 2.37 g of cobalt chloride hexahydrate, 1.185 g of nickel chloride hexahydrate and 2 g of urea were dissolved in 75 mL of Milli-Q water and stirred for 30 min, and then the beaker containing the growth solution was covered completely with aluminum foil and kept in a preheated oven at 95C for 5 h. Aer that NiCo2O4 nanostructures were taken out by

ltration from the growth solution and washed with Milli-Q water. In last step at room temperature sample were dried and additionally these nanostructures were annealed at 500C for 3 h to convert nickel cobalt hydroxide into nickel cobalt oxide phase.

10 mg of NiCo2O4nanoneedles were prepared in 2.5 mL of

Milli-Q water and sonicated for 15 min, aer that 500 mL of Naon (5%) was added in it, based on previous investigations.34

Then the GCE was polished with alumina powder, covered with

Table 1 Analytical application of NiCo2O4nanoneedle-modiﬁed/GCE

urea sensor for the quantiﬁcation of urea from diluted urine samples

Sample Added (mM) Detected (mM) Recovery (%)

1 0 0.28 0.03 0.5 0.50 0.03 100.7 1 0.95 0.04 95.2 1.5 1.52 0.21 101.6 2 0 0.28 0.01 0.5 0.52 0.05 104.8 1 0.96 0.03 96.3 1.5 1.45 0.36 97.1 3 0 0.15 0.48 0.5 0.49 0.22 99.2 1 1.04 0.04 104.7 1.5 1.52 0.59 101.7

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5mL of NiCo2O4nanoneedles with weight of 0.2 mg by drop

casting, dried for 10 min at room temperature, and then ready to be used for urea measurements. The presence of Naon guarantees the good adhesion of the nanoneedles to the GCE. Synthesis of NiO and Co3O4benchmarking electrodes

NiO nanostructures were synthesized by low temperature aqueous chemical growth method using a two-step approach. First, nickel hydroxide was obtained by mixing equi-molar 0.1 M concentration of nickel chloride hexahydrate and hexamethy-lenetetramine in a glass beaker of 100 mL capacity. The growth solution was covered with aluminum foil and kept in preheated electric oven at 95C for 5 hours. Aer the completion of the growth, the nickel hydroxide nanostructured material was ob-tained byltration and washed several times with deionized water and ethanol. Then nickel hydroxide nanostructures were calcinated at 450C for 3 hours in air and NiO nanostructured material was successfully obtained. Likewise, nanostructured Co3O4was prepared by the same two-step procedure, by using

diﬀerent precursors, i.e. 0.1 M CoCl2$6H2O and urea, under the

same experimental conditions described above. Morphology and structure

The shape evolution of NiCo2O4 nanostructures was

investi-gated by high resolution LEO 1550 Gemini eld emission scanning electron microscope working at 10 kV. The crystal quality was evaluated by X-ray powder diﬀraction (XRD) of Phillips PW 1729 powder diﬀractometer using CuKa radiation (l¼ 1.5418 ˚A), a generator voltage of 40 kV and a current of 45 mA.

HRTEM and STEM images have been obtained by using a FEI Tecnai F20eld emission gun microscope with a 0.19 nm point-to-point resolution at 200 kV equipped with an embedded Quantum Gatan Image Filter for EELS analyses. Images have been analyzed by means of Gatan kDigital Micrograph soware. The X-ray photoelectron spectroscopy (XPS) experiments were

carried out using a Scienta ESCA 200 spectrometer in ultrahigh vacuum at a base pressure of 1010mbar with a monochromatic Al (K alpha) X-ray source providing photons with 1486.6 eV. The XPS experimental condition was set so that the full width at half maximum of the clean Au 4f7/2line was 0.65 eV. All spectra were

collected at a photoelectron takeoﬀ angle of 0 _(normal

emis-sion) at room temperature. Functional characterization

All voltammetry measurements were carried out by using a CHI 760D Electrochemical Workstations. The soware CHI 9.22 (Austin, USA) was used in combination with the electrochemical workstation. A conventional assembly of three-electrode system was used. The maximum capacity of solution in cell was 5– 10 mL with gas bubbler and gas outlet ports.

GCE with a diameter of 2 mm used as working electrodes, in combination with platinum (Pt) wire as counter and Ag/AgCl as reference electrode (purchased from CHI Austin, USA) were used as received. The working electrodes were manually cleaned before each scan by mechanical polishing using alumina powder.

Solution of 0.1 M NaOH (E. Merck-Germany) was prepared in Milli-Q water and was used as supporting electrolyte. A stock solution of 100 mM urea was prepared in sodium hydroxide. The low concentration standard solutions of urea were freshly prepared before the measurements by dilution. Stock solution of Naon (5%) (Sigma Aldrich) was prepared in isopropanol. All the measurements were performed at room temperature. The peak current in cyclic voltammetry curve for urea oxidation was found around +0.75 V vs. Ag/AgCl reference electrode.

Results and discussion

Fig. 2 shows the SEM and ADF-STEM images of NiCo2O4

nanoneedles. These nanoneedles are formed by the self-assembly of nanoparticles. The EELS chemical composition

Fig. 1 Scheme for the fabrication of the non-enzymatic urea sensor based on NiCo2O4nanoneedle-modiﬁed GCE, from nanomaterials

synthesis, to sensor testing.

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maps for Co, Ni and O obtained from the ADF-STEM micro-graph are also reported. The compositional analysis (also conrmed in Fig. S1†) indicates that the nanoneedles are composed of evenly distributed Co, Ni and O, as expected. Fig. 3 (le panel) shows the TEM microscopy on a single particle, including its high resolution and FFT analysis. The FFT spec-trum indicates that the material crystallizes in the cubic Co3O4/

NiCo2O4 phase, [kFD3-MZ]-space group 227, with lattice

parameters a¼ b ¼ c ¼ 0.8065/0.8114 nm, and a ¼ b ¼ g ¼ 90 as visualized along the [0–11] direction. Further HRTEM anal-ysis revealed also the presence of cubic phase of NiO, as shown in Fig. S2.† The morphology and structure of the NiO and Co3O4

nanostructures are reported in Fig. S3–S7.† NiO presents a ower sheet-like structure (Fig. S3 and S4†) with a cubic

crystalline phase (Fig. S5†). The Co3O4presents a nanowire-like

structure, as visible in Fig. S6,† with a diﬀraction pattern ascribable to a cubic structure (Fig. S7†).

The EDS analysis (Fig. S8(a–c)†) indicated the expected composition for (a) NiO and (b) Co3O4, and (c) the presence of

20.5% in weight of Ni in the composite system, suggesting that, most probably, Ni is incorporated into the NiCo2O4phase.

The powder X-ray diﬀraction pattern (Fig. 3 (right panel)) conrmed the presence of reections from the cubic Co3O4

phase (JCPDS card no.¼ 42-1467) and cubic NiO phase (JCPDS card no.¼ 04-0835) in samples Co3O4and NiO, respectively, as

expected. Reections from the cubic Co3O4or NiCo2O4phase

are present in the composite system (NiCo2O4– JCPDS card no.

¼ 20-078 and Co3O4 cannot be discriminated through XRD), Fig. 2 (a) SEM and (b) ADF STEM images of NiCo2O4nanoneedles. (c) EELS elemental distribution maps obtained with the Co L2,3-edge (red), Ni

L2,3-edge (blue), O K-edge (green) and their color composite in NiCo2O4nanoneedles from the red squared area in (b).

Fig. 3 (Left) High resolution transmission electron microscopy of NiCo2O4nanoneedles and FFT spectrum. (Right) Powder XRD spectrum of

NiCo2O4nanoneedles at room temperature.

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together with two low-intensity reections (blue arrows), which suggest the presence of some NiO phase. From these analyses, we can conclude that the composite system is most likely composed of NiCo2O4cubic phase and some residual NiO cubic

phase.

The chemical composition of NiCo2O4 nanomaterial was

investigated by X-ray photoelectron microscopy (Fig. 4). The wide scan survey spectrum of the sample conrms the presence of Co, Ni and O. The photoemission peak at binding energy of 780.4 eV corresponds to Co 2p3/2and the peak at the binding

energy of 796.2 eV corresponds to Co 2p1/2. Satellite peaks at

786.7 and 802.5 eV binding energy are assigned to Co 2p3/2and

to Co 2p1/2, respectively, in agreement with the previous

litera-ture. The Ni 2p3/2peak at 855.1 eV and Ni 2p1/2peak at 873.2 eV

are also present, indicating the presence of NiO.35

The nanoneedle/GCE electrode was used for urea detection in 0.1 M NaOH solution containing 0.1 M of urea (Fig. 5). We also tested bare GCE without nanoneedles both in pure elec-trolyte (black curve) and in presence of 0.1 M urea (red curve). In the inset of Fig. 5(a) we report the response curve for the nanoneedle/GCE in 0.1 M NaOH. When NiCo2O4nanoneedle/

GCE is used, two clear oxidation and reduction peaks were recorded: one anodic peak at around 750 mV and one cathodic peak at around 550 mV. The analysis of the oxidation/reduction peaks ratio and of the peak to peak separation, as a function of the scan rate (Fig. S13 and Table S2†), recorded at diﬀerent scan rates, seems to indicate that oxidation/reduction or urea follow a semi-reversible process. It is because of the weak urea adsorption prior to oxidation at the proposed NiCo2O4

nano-needle. On the other end, a very weak oxidation signal was recorded, at the bare GCE without nanoneedles (Fig. 5(a)). The results conrm the electro catalytic properties of NiCo2O4

nanoneedle towards urea.

The proposed enzyme free sensing mechanism for the alkaline oxidation of urea is described as follows, aer the immediate absorption of OH-ions, both the Ni2+and Co2+are oxidized into Ni3+ _{and Co}3+_{. Then, both the Ni}2+_{and Co}2+_are

oxidized into Ni3+_{and Co}3+_{. Moreover, the alkaline mechanism}

is according to the reported work:36_{later the urea is adsorbed on}

the NiOOH by Ni–O and O–C coordinate linkages and simul-taneous direct oxidation of urea is taking place on NiOOH.37

Furthermore, the NiOOH is reduced to Ni(OH)2at the time of

urea oxidation. The reported work suggests that cobalt ions are also involved in the oxidation of urea which is poor in response towards urea oxidation that is mainly coming from the presence of Co4+/Co3+active sites.38

The cyclic voltammograms of the NiCo2O4nanoneedles/GCE

in 0.1 M urea solution recorded at diﬀerent scan rates (between 10 and 100 mV s1) are reported as an inset in Fig. 5(b). The plot of peak current of urea oxidation has a linear dependence upon the square root of scan rate as shown in Fig. 5(b) indicating the diﬀusion control of the reaction.

To demonstrate the stability of the NiCo2O4nanoneedle and

the reproducibility of the measurements, we recorded 16 repetitive runs for 0.1 M of urea (Fig. 6(a)). The relative standard deviation (RSD) for the obtained results was 4%. These results conrm the stability, upon use, of the NiCo2O4 nanoneedles/

GCE electrode and the reproducibility of its electro-catalytic performance over urea oxidation. The specicity of the NiCo2O4nanoneedle/GCE electrode to urea was tested toward

likely interferences including glucose, ascorbic acid, uric acid and their mixtures. Cyclic voltammetry experiments were carried out for 0.1 M of urea in the presence of equi-molar concentration of each interferent and mixtures of them. Fig. 6(b) represents some of the potential interferents response during the sensing of urea. The response of urea at NiCo2O4

Fig. 4 (a) Wide scan survey XPS spectrum of NiCo2O4nanoneedles. (b) Ni 2p spectrum. (c) Co 2p spectrum.

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nanoneedle/GCE is not aﬀected by the presence of interferents such as glucose, uric acid and ascorbic acid, demonstrating that the sensor is suitable for selective urea detection in real applications.

The calibration curve at various urea concentrations (Fig. 6(c)) indicates the linear dependence of the recorded current vs. diﬀerent urea concentrations in the range 0.01 to 5 mM inset in (Fig. 6(c)) shows peak current vs. urea concen-tration range from 0.01 to 1 mM. The resulting R2value is 0.99, which indicates a good linear behavior of the electrode in the specied concentration range. The corresponding CV runs at diﬀerent concentrations are enclosed in the ESI (Fig. S9†).

The lower detection limit (LOD) and quantication limit (LOQ) of the proposed sensor were calculated36_{as DL/DQ}_{¼ F}

SD/b, where the factor F is equal to 3.3 and 10 for the detection limit and the limit of quantication, respectively, SD is the standard deviation of the blank, and b is the slope of the linear regression of the calibration curve. The LOD was calculated to be 1.0mg cm3and the LOQ was calculated to be 3.6mg cm1.39

Table S3† collects the comparative data from the literature on various methods and materials used for urea sensing. The overall performance of the NiCo2O4 nanoneedle/GCE is

competitive or even far better than the previously reported sensors. As reported in Table S3,† most of the sensors are urease-based, which makes the system complex and expensive. Additionally, the detection limit of the newly prepared electrode was outstandingly lower than other reported values, represent-ing a signicant advancement in the specic eld and makrepresent-ing the new nanoneedle/GCE electrode a viable candidate for practical use. For the demonstration of the outstanding prop-erties of the NiCo2O4nanoneedles, a comparative CV study was

performed under the same experimental conditions, applying NiO and Co3O4nanostructures for urea sensing (Fig. S10†). The

oxidation peak current in the case of nickel cobalt oxide

nanoneedles is highly enhanced compared to both NiO and Co3O4, which can be attributed to the excellent electrocatalytic

properties of the bimetallic oxides. This is further veried by electrochemical impedance analysis. The Nyquist plot obtained in absence of any faradaic process (+200 mV) was employed for the determination of the double layer capacitance of the elec-trode materials as shown in Fig. S11,† as described earlier.40_The

CDLvalues obtained are respectively 0.038 mF cm2 for NiO,

0.044 mF cm2for Co3O4and 0.059 mF cm2for NiCo2O4. The

higher CDL value for the bimetallic oxide suggests a larger

number of active sites due to the larger electrochemical surface area (assuming comparable specic capacitance). Therefore, we chose to be more conservative and only qualitatively compare the variation of double layer capacitance.41_{The EIS method has}

been proved to be highly reliable and fully consistent with the CV method, as previously reported.40

The developed sensor was successfully applied to the anal-ysis of urine samples. Urine samples were collected from three healthier people (lab mates) aer 2 h of their breakfast. Prior to analysis, urine samples wereltered through a 3 mm size of lter paper, to get rid of protein aggregates. The urine samples were diluted in NaOH (supporting electrolyte) at the ratio of 1 : 10 by volume; they were then spiked with known concentrations of urea solutions. The modied electrode was used to analyze three urine samples. The inuence of the matrix was examined by performing recovery experiments and the results were esti-mated from the calibration curve. All experiments were per-formed three times, to guarantee their reproducibility. Recoveries and % RSD values of each sample are given in Table 1. The CV runs for the determination of urea from real samples are provided in the ESI (Fig. S12†), demonstrating the practical application of the proposed sensor for urea monitoring in real urine samples.

Fig. 5 (a) CV response of bare GCE (red) and NiCo2O4nanoneedle/GCE (blue) in 0.1 M urea, and response of bare GCE in blank 0.1 M NaOH

(black). Inset: nanoneedle/GCE in 0.1 M NaOH. (b) Anodic peak current of various CV runs versus the square root of the scan rate. Inset: CV response of the NiCo2O4nanoneedle/GCE electrode in 1 mM urea at diﬀerent scan rates.

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Conclusions

In summary, we proposed bimetallic oxide nanoneedles of NiCo2O4, synthesized by low temperature aqueous chemical

growth method, as new enzyme-free urea sensors. The high purity NiCo2O4 nanoneedles result from the self-assembly of

nanoparticles and exhibit cubic crystalline phase. Owing to their enhanced catalytic activity and conductivity, application of NiCo2O4nanoneedles was found highly eﬀective for the

selec-tive and sensiselec-tive quantication of urea both in chemical sensing and urine samples.

The superior performance of NiCo2O4nanoneedle-modied/

GCE non-enzymatic urea sensor over urease immobilized elec-trode matrix biosensors can be used towards the development of non-enzymatic urea sensing. Urease-based biosensors are very selective and sensitive, however they present signicant

drawbacks, including use of expensive urease enzyme (and need of specic conditions for storage), thermal stability, sensitivity to harsh acidic and alkaline media,42_{and loss of enzyme activity}

with time39 _{The proposed NiCo}

2O4 nanoneedle-modied/GCE

non-enzymatic urea sensor is highly selective, sensitive and stable, compared to the urease based biosensors, exhibiting the lowest detection limit ever reported, equal to 1.0mM. The pre-sented protocol for urea measurement has a series of merits compared to the reported literature such as easy accessibility in harsh conditions, including high pH, wide linear range of urea, high reproducibility, facile fabrication process, and does not require specic storage conditions. The NiCo2O4nanoneedles

are obtained from earth abundant and cheap sources, making them economically viable in both developed and developing countries. The presented urea sensor is enzyme-free, making it a unique conguration, suitable for large scale applications.

Fig. 6 (a) Sixteen repeated CVs measurements of the NiCo2O4nanoneedle-modiﬁed/GCE sensor in 0.1 M urea to monitor the stability and

reproducibility. (b) Inﬂuence of potential interferents on the voltammetry response in 0.1 M urea. (c) Calibration plot (peak current versus urea concentration) in the concentration range 0.01–5 mM.

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We also demonstrated the successfully use of the sensor for the monitoring of urea from urine samples. Our results indicate that the NiCo2O4nanoneedle-based urea sensor can be

capi-talized for routine analysis of urea from various clinical and food samples and represents a signicant advancement in the eld.

Ethical statement

Experiments related to urine analysis were approved by the ethics committee at Sindh University Jamshoro, Sindh Pakistan. Informed consents were obtained from human participants of this study.

Con

ﬂicts of interest

Authors declare no conict of interest in this research work.

Acknowledgements

S. A. acknowledges the Shaheed Benazir Bhutto University, Shaheed Benazir abad fornancial support during the study visit at the Lule˚a University of Technology Sweden as part of her PhD program. A. V. acknowledges the European Commission under grant agreement GA 654002, the Wallen-berg Foundation, the Swedish Foundations, the Kempe Foundation, the LTU Lab fund program and the LTU Seed project for partial funding. The authors are grateful to Prof. I. Lundstr¨om for his suggestions and constructive discussions during the preparation of the manuscript. ICN2 and IREC acknowledge funding from Generalitat de Catalunya 2014 SGR 1638 and the Spanish MINECO coordinated projects TNT-FUELS and e-TNT (MAT2014-59961-C2). ICN2 acknowledges support from the Severo Ochoa Programme (MINECO, Grant no. SEV-2013-0295) and is funded by the CERCA Programme/ Generalitat de Catalunya. Part of the present work has been performed in the framework of UniversitatAut`onoma de Bar-celona Materials Science PhD program.

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